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Tlr3 Activation Links Damage Sensing To Epimorphic Regeneration In The Skin
Sashank K. Reddy, MD, PhD1, Amanda Nelson, PhD2, Emily Chang, BA1, Tabetha Ratliff, MA1, Adiya Katseff, BA1, Sydney Resnik, BA1, Lloyd S. Miller, MD, PhD1, Luis A. Garza, MD, PhD1.
1Johns Hopkins University, Baltimore, MD, USA, 2Pennsylvania State University, Hershey, PA, USA.
PURPOSE: Animals across diverse phyla can regenerate lost structures, a capacity that is considerably more limited in humans and other mammals. An exception is wound-induced hair neogenesis (WIHN), a recently reported phenomenon in which skin and hair follicles are regenerated following full thickness wounds in mice. Hair follicles are complex mini-organs with disparate cell types, dedicated neurovascular support, and a distinct stem cell compartment. These stem cells not only repopulate hair follicles, but also aid in skin re-epithelialization after wounding. Further, as WIHN represents a rare example of adult organogenesis in mammals, understanding its mechanisms could aid in efforts to regenerate other structures. Here we describe the role of the innate immune receptor TLR3 in linking damage sensing after wounding to the earliest molecular events in hair regeneration.
METHODS: WIHN assays were performed following full thickness wounding on the backs of mice. Regenerated hair numbers were quantified by confocal scanning laser microscopy or immunohistochemistry. Microarray analyses were performed on reverse transcribed mRNA harvested from scars of highly regenerating and poorly regenerating mouse strains at the time of wound closure. Keratinocytes in culture were treated with the TLR3 agonist poly-inosine-cytosine (PIC). Quantitative PCR experiments were performed on reverse transcribed mRNA using the TaqMan system. In statistical comparisons, differences were considered significant at p <0.05.
RESULTS: Microarray analyses revealed that innate immune signaling was highly upregulated in mice with a high regenerative capacity. Specifically, mRNA for TLR3, an innate immune receptor activated by dsRNA, was significantly increased in highly regenerative mice. This observation was functionally validated in TLR3 -/- mice which had greatly reduced regeneration compared to strain matched controls (0 follicles vs. 5.5 follicles). Addition of PIC – a TLR3 agonist and dsRNA mimic – increased the extent of hair regeneration in mice (25 follicles vs. 5 follicles), whereas inhibition of dsRNA using RNAse III decreased regeneration (2 follicles vs. 4 follicles). The effects of TLR3 on hair regeneration were dependent on the downstream target IL6 and its corresponding transcription factor STAT3. In vitro, TLR3 activation prevented keratinocyte differentiation and led to upregulation of the keratinocyte stem cell genes TAp63 and CBX4. TLR3 signaling also led to the activation of the core developmental program for hair follicle morphogenesis including the Wnt and Shh pathways.
CONCLUSIONS: Wound-induced hair neogenesis represents a rare example of adult regeneration and organogenesis in mammals. This study reveals a novel and important interplay between innate immune activation and regeneration. Specifically, TLR3 is activated by molecules released during tissue damage such as dsRNA and promotes activation of core developmental pathways for regeneration in the skin. Stimulation of TLR3 thorough synthetic ligands may lead to improved cutaneous wound healing and hair follicle regeneration.
Figure 1. The TLR3 agonist PIC promotes hair follicle neogenesis.
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